1. Field of the Invention
The present invention relates to the field of molecular genetics and medical diagnosis. More specifically, the invention provides a method for detecting chimeric mRNA sequences containing a specific exon-exon junction by reverse transcription into complementary DNA (cDNA), amplification of the cDNA sequences by the Polymerase Chain Reaction (PCR), and analysis of the amplified material, if any. The method is especially useful for the detection as well as analysis of unique chimeric mRNA sequences indicative of normal or aberrant cell types, chimeric mRNA associated with certain stages of cellular development, and chimeric mRNA sequences associated with oncogenesis.
2. Description of the Related Art
This application describes methods of genetic analysis that utilize the polymerase chain reaction described in copending Ser. No. 063,647, filed June 17, 1987, which is a continuation-in-part of Ser. No. 899,513, filed Aug. 22, 1986, now abandoned, which is a continuation-in-part of Ser. No. 828,144, filed Feb. 7, 1986, which issued as U.S. Pat. No. 4,683,195, and which is a continuation-in-part of Ser. No. 791,308, filed Oct. 25, 1985, which issued as U.S. Pat. No. 4,683,202, and which is a continuation-in-part of now abandoned Ser. No. 716,975, filed Mar. 28, 1985. The polymerase chain reaction can employ a thermostable polymerase described in Ser. No. 143,441, filed Jan. 12, 1988, which is a continuation-in-part of copending Ser. No. 063,509, filed June 17, 1987, which issued as U.S. Pat. No. 4,889,818, which is a continuation-in-part of Ser. No. 899,241, filed Aug. 22, 1986, now abandoned. The polymerase chain reaction has been automated; an apparatus capable of carrying out the reaction is disclosed in related copending Ser. No. 899,061, filed Aug. 22, 1986, which is a continuation-in-part of Ser. No. 833,368, filed Feb. 25, 1986, now abandoned. The present invention also provides methods of dot-blot hybridization and so is related to copending Ser. No. 197,000, filed May 20, 1988, which is a continuation-in-part of Ser. No. 899,344, filed Aug. 22, 1986, now abandoned, which is a continuation-in-part of Ser. No. 839,331, filed Mar. 13, 1986, which is now abandoned. In addition, the invention discloses methods for using enzyme labeled oligonucleotide probes and so is related to Ser. No. 103,978, now abandoned in favor of continuation Ser. No. 437,311, filed Oct. 2, 1987, and is related to Ser. No. 104,200, filed Oct. 2, 1987, which has issued as U.S. Pat. No. 4,914,210. The disclosure of these related applications and patents are incorporated herein by reference.
The techniques of molecular biology have yielded new insight about disease processes at the molecular level. Gene cloning has made it possible to understand genetic fine structure and the mechanisms of gene regulation. As a result, the aberrant effects of genetic alteration, i.e. mutation, deletion, insertion, substitution or amplification are becoming better characterized. In turn, the techniques of molecular biology have moved from basic research to applied medicine with the development of new diagnostic tools for improved prognosis and treatment.
The detection of mRNA expressed from a particular gene is one approach in determining a gene's activity. The detection of mRNA is done primarily through the use of labeled hybridization probes. See, for example, Maniatis, T., et al. Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory (1982). The hybridization can be done in solution or the RNA can be immobilized onto a membrane and subsequently detected as in the Northern format and RNA dot blot hybridizations. Traditional detection methods are limited by requiring large amounts of mRNA. If the mRNA of interest is present in low copy number, large amounts of biological material will need to be extracted and the mRNA enriched by such methods as density gradient centrifugation and oligo (dT)-cellulose chromatography. In addition, detection of mRNA sequences can be achieved by reverse transcription of the mRNA into cDNA and detection of the cDNA directly with a labeled hybridization probe. The cDNA can also be cloned into a plasmid and detected by colony hybridization. For diagnostic purposes, however, such methods for detecting mRNA sequences are impractical due to the cost of time, labor, and materials.
In recent years, the correlation of gene activity with disease state has focused on the association between proto-oncogene expression and the events of carcinogenesis. For example, Slamon, D. J., et al. Science 224: 256-262 (1984), demonstrated the expression of 15 proto-oncogenes by Northern analysis of RNA from fresh human tumors from 54 patients, representing 20 different tumor types. The study required the extraction of large tissue samples to obtain the 5 .mu.g of RNA needed for the analysis.
Similar studies of proto-oncogene expression have shown the association between overexpression of a variety of genes and cancers, including: the neu/HER-2 mRNA and breast cancer, Van De Vijver, M. et al., Mol. Cell. Biol. 7: 2019-2023 (1987); c-MYC mRNA and breast cancer, Escot, C., et al. PNAS 83: 4834-4838 (1986); c-MYC mRNA in fresh tumor material from patients with hematopoietic malignancies, Rothberg, P. G. Mol. Cell. Biol. 4: 1096-1103 (1984); H-ras mRNA and breast cancer, Theillet, C., et al. Cancer Res. 46: 4776-4781 (1986); N-myc mRNA and neuroblastoma, Michitsch, R. W., et al. Mol. Cell. Biol. 4: 2370-2380, (1984); c-MYC mRNA and cervical cancer, Riou, G., et al. Lancet 1: 761-763 (1987); and c-, N-, or L-MYC mRNA and small cell lung cancer (SCLC) cell lines, Nau, M. M., et al. Nature 318: 69-73 (1985). In each case, large sample specimens had to be extracted to obtain sufficient amounts of mRNA for the analyses. Such analyses would be impossible with small samples taken for early diagnosis when tumor mass is barely discernable.
Genetic rearrangements such as chromosome translocation can also result in the expression of proto-oncogene mRNA. For example, Philadelphia chromosome (Ph.sup.1, t[9;22] [q34; q11]) associated with chronic myeloid leukemia (CML) is a translocation that results in the combining of sites near or within the proto-oncogene c-ABL with a region of chromosome 22 called the breakpoint cluster region gene (BCR). The BCR-ABL sequence has been shown by Shtivelman, E. et al. Cell 47: 277-284 (1986) to express an 8.7 kb mRNA. The RNA analysis methods used, e.g., Northern blotting, S-1 mapping and RNAase protection, required large amounts of RNA and several days to complete. Such methods are impractical as diagnostic tools. U.S. Pat. No. 4,681,840 issued July 21, 1987, discloses single-stranded DNA probes for detecting BCR sequences and associated chromosome translocations.
In a similar manner, Northern blotting and S-1 mapping were used by Leder, P., et al. Science 222: 765-771 (1983), to show the disregulation of c-MYC expression from chromosome 8q24 in Burkitt's lymphoma. The rearrangement combines the c-MYC gene with one of three immunoglobulin loci on chromosomes 14(q32), 2(q13) or 22(q11), as described by Liein, G., Cell 32: 311 (1983).
RNA expression levels of the proto-oncogenes c-MYC, c-FOS, and c-FMS, were measured in bone marrow cells obtained from patients with acute myeloid leukemia (AML), Preisler, H. D., et al. Cancer Res. 47: 847-880 (1987). The results suggested the possible use of proto-oncogene expression patterns as a means of more accurately categorizing leukemic cell subtypes. The elevated expression of proto-oncogene mRNA has in some instances been found to be associated with chromosome deletion. For example, Barletta, C., et al. Science 235: 1064-1067 (1987), show that amplification and overexpression of the c-MYC locus accompanies deletion of the long arm of chromosome 6 and suggest an involvement in the pathogenesis of leukemias and lymphomas.
PCR amplification followed by oligonucleotide dot blot analysis was used to study RAS gene mutations in acute myeloid leukemia (AML). The study screened for point mutations in codons 12, 13, and 61 and found 27% of the patients contained mutations which were predominantly in codon 12, Farr, C. J., et al. PNAS 85: 1629-1633.
The use of RNA dot-blot hybridization is a common method to measure gene expression. Pinto, A., et al. Blood 70: 1450-1457 (1987), applied RNA dot-blot techniques to measure c-FOS oncogene expression in human hematopoietic malignancies. The method required isolation of the leukemic cells before RNA extraction, and large quantities of RNA were required for detection and quantitation of proto-oncogene expression.
Hybridization to DNA sequences is the primary approach for detecting altered gene structure. DNA hybridization lacks the sensitivity needed to discriminate diseased from healthy cells in the early stages of illness, e.g., for cancer diagnosis. Yoffe, G., et al. Blood 69: 961 (1987), showed 5% of a patient's lymphocytes must be leukemic before deletions and translocations in genomic DNA can be detected by Southern transfer. Although hybridization with DNA sequences allows detection of genomic fine structure and chromosomal rearrangement, the method indicates nothing about gene activity.
U.S. Pat. No. 4,683,202, incorporated herein by reference, discloses a method for amplifying specific nucleic acid sequences, called the Polymerase Chain Reaction (PCR). U.S. Pat. No. 4,683,195, incorporated herein by reference, discloses a method of using PCR for amplifying, detecting and cloning nucleic acid sequences. PCR causes the exponential amplification of target nucleic acid sequences from small amounts of material. The PCR process utilizes temperature regulated cycling of oligonucleotide primer hybridization and DNA polymerase mediated synthesis of target sequences from the hybridized primers. Because thermal denaturation of polymerase results in decreased amplification, the PCR format has been greatly simplified through the use of the thermostable DNA polymerase (Taq) isolated from the thermophilic bacterium, Thermus aquaticus. A process for amplifying, detecting, and cloning nucleic acid sequences using a thermostable enzyme is disclosed in U.S. application Ser. No. 063,647, filed June 17, 1987, incorporated herein by reference.
The isolation, cloning and physical properties of the Taq enzyme, together with stable enzyme compositions having a purified, thermostable polymerase enzyme such as Taq in a buffer containing one or more non-ionic detergents are disclosed in U.S. application Ser. No. 063,509, filed June 17, 1987, incorporated herein by reference. The PCR amplification of translocated DNA sequences requires knowledge of the translocation junction. This is difficult when the translocation occurs randomly within long segments of DNA. For example, Groffen, J., et. al. Cell 36: 93-99 (1984), incorporated herein by reference, found that in 17 of 17 Ph.sup.1 positive CML patients, the BCR breakpoint occurred within 5.8 kb, while the c-ABL breakpoint was dispersed along 100 kb.
The PCR amplification and analysis of mRNA via cDNA with a thermostable enzyme has been shown in commonly owned and copending U.S. patent application Ser. No. 899,513, filed Aug. 22, 1986. Powell, L. M., et al. Cell 50: 831-840 (1987), have applied PCR amplification of cDNA to the study of a novel form of tissue-specific RNA processing for apolipoprotein-B48 in the intestine. In addition, PCR amplification of double stranded cDNA has been used by Todd, J. A., et al. Nature 329: 599-604, to facilitate the cloning of the gene for HLA-DQ.beta..